The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are considered: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs has cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg, J. et al., Lancet, 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001)). Numerous other studies document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
A variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Genetic engineering has demonstrated that the natural abilities of some hosts can be substantially altered to produce various long-chain ω-3/ω-6 PUFAs, e.g., arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3). Whether ω-3/ω-6 PUFA production is the result of natural abilities or recombinant technology, however, both strategies may benefit from methods that increase carbon flow into lipid metabolism.
Lipid metabolism in most organisms is catalyzed by a multi-enzyme fatty acid synthase (FAS) complex and initially occurs by the condensation of eight 2-carbon fragments (acetyl groups from acetyl-CoA) to form palmitate, a 16-carbon saturated fatty acid (Smith, S. FASEB J., 8(15):1248-59 (1994)). Once free palmitate (16:0) is released from FAS, the molecule undergoes either elongation (i.e., via a C16/18 fatty acid elongase to produce stearic acid (18:0)) or unsaturation (i.e., via a Δ9 desaturase to produce palmitoleic acid (16:1)). The primary fate of palmitate is elongation, while desaturation is only a minor reaction in most organisms. As such, significant cellular pools of stearic acid become available for conversion to oleic acid (18:1) via a Δ9 desaturase. Oleic acid is the primary metabolic precursor of all other fatty acid molecules. Based on the above, it is concluded that Δ9 desaturases affect overall carbon flux into the fatty acid biosynthetic pathway, and thereby play a determinant role in both the quantity and composition of oil so produced.
Based on the role Δ9 desaturase enzymes play to thereby effectively “push” carbon into the PUFA biosynthetic pathway, there has been considerable effort to identify and characterize these enzymes from various sources. For example, the genes encoding Δ9 desaturase have been cloned from several fungi and yeast, including: Saccharomyces cerevisiae (Stukey J. E. et al., J Biol. Chem., 265(33):20144-20149 (1990)); the oleaginous yeast Cryptococcus curvatus CBS 570 (Meesters, P. A. and G. Eggink, Yeast, 12(8):723-730 (1996)); Saccharomyces kluyveri (GenBank Accession No. AB071696; Kajiwara S., FEMS Yeast Res., 2(3):333-339 (2002)); Mortierella alpina (U.S. Pat. No. 6,448,055); and Aspergillis nidulans (U.S. Pat. No. 6,495,738). And, expression of some of these Δ9 desaturases in non-native host organisms has been shown to increase the level of palmitoleic acid, oleic acid and derivatives thereof (e.g., U.S. Pat. No. 6,448,055). Despite this, there is need for the identification and isolation of additional genes encoding Δ9 desaturases that will be suitable for heterologous expression in a variety of host organisms for use in the production of PUFAs.
There are no reports to date concerning the isolation of a Δ9 desaturase from an euglenoid. Although there are over 100 species described within the genus of euglenoids known as Euglena, E. gracilis is best studied. Several other investigators have studied the PUFA biosynthetic pathway within this organism, leading to the isolation of the organism's Δ8 desaturase and Δ4 desaturase; however, no one has identified the gene encoding Δ9 desaturase within Euglena gracilis. 
Applicants have solved the stated problem by isolating the gene encoding Δ9 desaturase from Euglena gracilis and demonstrating increased conversion of 18:0 to 18:1 upon over-expression of the gene in the oleaginous yeast, Yarrowia lipolytica. This will enable increased PUFA content in the host oil and increased oil biosynthesis, upon co-expression with other PUFA biosynthetic pathway genes.